Since the genomic analysis has been completed and all of the expressed genes are being elucidated, the importance of the functional analysis of proteins has increased. In addition, owing to the development of molecular biology, it has become possible to make a large quantity of proteins express as recombinant proteins by means of introducing recombinant genes into the cells of animals and plants, yeasts, bacteria, and the like, in place of purification of proteins from living tissues.
In order to obtain a recombinant protein, however, it is necessary to separate and purify an aimed recombinant protein from a large quantity of cell-derived impurities such as proteins, nucleic acids, polysaccharides and so on, after expression of the recombinant protein and homogenate of the cells in a general way. In the production of recombinant proteins using cells as host, accordingly, it is a very important issue for the functional analysis and utilization of proteins to carry out the separation and purification of an aimed recombinant protein efficiently.
A frequently-used effective method for separating and purifying recombinant proteins is affinity chromatography. In this method, a gene sequence coding for the aimed protein and another gene sequence coding for a protein fragment having a high affinity to a certain ligand (hereinafter, referred to as affinity peptide) are utilized to make express a recombinant fused protein in which the aimed protein is combined with the affinity peptide. Subsequently, this recombinant fused protein is isolated from other impurities by using a carrier on which the ligand binding to the fused affinity peptide has been immobilized, and then the affinity peptide is dissociated therefrom. Thus, the aimed peptide can conveniently be purified. In practice, there are some procedures known as affinity chromatography, i.e., procedure using polyhistidine (Patent document 1), procedure using glutathione-S-transferase (GST) (Patent document 2), and procedure utilizing a maltose-binding protein (MBP).
However, there were some problems inmost of the affinity chromatography which had been developed in the past. For example, some recombinant fused proteins having a combination of polyhistidine and an aimed protein were often insolublized by a host or did not show an inherent activity. In particular, when a huge affinity peptide having the molecular weight of about 30-50 kDa such as GST or MBP was fused, they were sometimes expressed as a recombinant fused protein having no inherent activity because their normal folding was obstructed. Further, there was an economic problem since a highly expensive ligand such as IgG antibody had to be used as a carrier for affinity chromatography. Additionally, for some affinity peptides, the recombinant fused proteins had to be eluted from the carrier at a pH value which may inactivate the aimed protein, or it was necessary to add a substance such as guanidine or ethylene glycol, which worked to denature the aimed protein or inhibit the activity. That is, affinity chromatography may have a practical problem that dissociation was difficult under a mild condition, though it enables highly specific purification by utilizing an fused affinity peptide and a ligand corresponding thereto.
In view of this situation, the present inventors have reported that a recombinant fused protein can be separated and purified in a relatively mild condition by utilizing dockerin, a protein which is a part of a protein complex termed cellulosome originating from Clostridium josui (reported by Ohmiya et al. in Non-patent document 1) and containing a calcium-binding motif domain, and another protein cohesin domain specifically binding via calcium ion (Patent document 3).
In this method for purification, however, there is a problem that when the recombinant fused protein is eluted at a low temperature around 4° C. in order to suppress denaturation of the protein, it takes 6 to 16 hours to elute the protein because the binding of dockerin to cohesin domain is strong. On the other hand, the protein can be eluted within a short period of time at ordinary temperature, but in such a case, there is concern that the activity of protein might be affected.    Patent document 1: Japanese Patent No. 2686090    Patent document 2: JP-A-H07-184663A    Patent document 3: WO 03/033695 International Publication Pamphlet    Non-patent document 1: The bulletin of the Faculty of Bioresource, Mie University, No. 19, pp 71-96 (1997)